.. In the course of studying inflammatory muscle diseases, we have encountered patients with other muscle diseases. We have studied patients with two genetic metabolic myopathies in detail: phosphofructokinase (PFK) deficiency, and Type II Glycogen Storage Disease (acid alpha-glucosidase deficiency - also known as acid maltase deficiency or Pompe Disease). Our experience of treating Pompe mice (a knockout strain) and the experience in trials in humans, carried out in part under a CRADA with Genzyme, the company producing the recombinant drug, demonstrated that the drug is highly effective in clearing glycogen stored in cardiac muscle cells and only poorly effective for skeletal muscle cells. This failure of skeletal muscle response to recombinant enzyme in Pompe Syndrome has become a major focus of our attention and has led us into active new areas of cell biology that in themselves add great interest to our activities. We noted remarkable differences between Type I &II Pompe in the distribution of lysosomes: in Type I fibers, lysosomes are arranged in long stretches leading to a tube-like structure, and Golgi markers neighbor lysosomes in Pompe as in WT fibers. By contrast, in Type II Pompe fibers, lysosomes are distributed throughout the fiber, and the Golgi markers are less regular and less likely to neighbor lysosomes. Most strikingly, the large areas of autophagic build-up, which may occupy close to half the diameter of a fiber, are usually centrally located, and disrupt the microtubular network and the contractile apparatus itself. With the Light Imaging Section and using second harmonic generation combined with two-photon excited fluorescence (E. Ralston and K. Zaal) we have noted early accumulation of lipofuscin in autophagic areas, presumably reflecting oxidative stress. The disturbed clearance of autophagic debris in fast fibers was accompanied by clear evidence in fast fibers of active autophagy. To study this we made autophagy-deficient Pompe mice. Muscle muscle-specific autophagy-deficient GAA-/- mice: MLCcre: Atg7F/F: GAA-/- were created by first crossing Atg7- conditional knockout mice to a skeletal muscle specific Cre line to generate muscle-specific autophagy-deficient mice (MLCcre: Atg7F/F: wt). These mice were crossed to GAA-/- mice to produce GAA-/- mice: MLCcre: Atg7F/F: GAA-/- . The need for tissue-specific suppression of autophagy is justified by the fact that mice with a global deletion of autophagic genes (Atg5 or Atg7) die soon after birth. The clinical signs of muscle disease were monitored in approximately 100 mice. The production of Atg5-deficient GAA-/- mice has been published. The white part of gastrocnemius and psoas muscles in mice are a good source of glycolytic fast-twitch type II fibers (referred to as fast fibers), whereas soleus muscle is a good source of oxidative slow-twitch type I fibers (referred to as slow fibers). At least 3 animals from each genotype were used to obtain single muscle fibers for immunostaining. For each immunostaining and for confocal analysis, at least 20 fibers were isolated from each of the three muscle groups. Immunostaining, confocal microscopy, Western blotting, glycogen measurement as previously described. For studies of enzyme replacement therapy (ERT), two and a half month-old GAA -/-, MLCcre: Atg7F/F: GAA-/-, and HSAcre: Atg5F/F: GAA-/- mice received 3 intravenous injections of recombinant human -glucosidase at a dose of 100 mg/kg every other week and the mice were sacrificed 7 days after the last injection. As expected, autophagy was completely suppressed in fast (gastrocnemius) but not slow (soleus) muscles in the MLCcre: Atg7F/F: GAA-/- mice as shown by the absence of LC3II, a highly specific marker for autophagic vesicles, called autophagosomes. Expansion of lysosomes, a hallmark of Pompe disease, persists in muscle fibers from MLCcre: Atg7F/F: GAA-/- mice, but autophagic accumulation is absent. A prominent feature of MLCcre: Atg7F/F: GAA-/- mice is that they show an age-dependent accumulation of ubiquitinated proteins in their skeletal muscles, suggesting a functional impairment of the lysosomes. The characteristics described above are similar to those seen in the previously described HSAcre: Atg5F/F: GAA-/- mice. The level of glycogen in MLCcre: Atg7F/F: GAA-/- was lower than in the GAA-/- by 57%, suggesting that autophagy may play a role in the delivery of lysosomal glycogen in this autophagy-deficient strain and clinically, the MLCcre: Atg7F/F: GAA-/- mice are less affected than the HSAcre: Atg5F/F: GAA-/-, and they appear to be no worse than the GAA-/-, if not better. Considering a low glycogen load in MLCcre: Atg7F/F: GAA-/- mice and the lack of additional clinical manifestations when compared to GAA-/-, these mice were good candidates for enzyme replacement therapy (ERT). Injection of the recombinant enzyme in these mice resulted in a dramatic reduction in the glycogen level approaching wild type levels This glycogen clearance was also demonstrated by PAS staining of muscle biopsies and by immunostaining of isolated single fibers for autophagosomal and lysosomal markers. In contrast, GAA-/- mice with genetically intact autophagy cleared glycogen poorly. The same ERT regimen in the HSAcre: Atg5F/F: GAA-/- strain with higher initial glycogen levels similarly led to complete removal of the accumulated glycogen. The results suggest that the removal of autophagic buildup is a major factor that causes this therapy to be so effective. Furthermore. ERT-treatment in autophagy-deficient GAA-/- strains resulted in a significant decrease in the amount of ubiquitinated-proteins in both soluble and non-soluble fractions. Showing that that the lysosomal function in treated autophagy-deficient GAA-/- mice is largely restored. It should be noted that even the most successful reversal of lysosomal pathology in ERT-treated autophagy-deficient GAA-/- mice leaves these animals autophagy-deficient in skeletal muscle. Our observational data (up to 18 months) have shown that skeletal muscle-specific suppression of autophagy in the wild type mice does not result in major abnormalities as shown by apparent strength, mobility, weight, and lifespan. Thus, the suppression of autophagy in skeletal muscle greatly facilitates the effect of ERT resulting in an outcome which has never been observed in Pompe mice with genetically intact autophagy. In the past year, we have successfully developed long-term lines of Pompe myoblasts transfected with CDK4 that retain the ability to mature and fuse into myotubes. Using these myotubes treated with RNAi for the autophagic protein atg 7 and using primary myotubes cultured from Pompe mice with knockout in skeletal muscle of the autophagic protein atg 5, we have noted that glycogen accumulation in lysosomes is unimpaired, clearly showing that macroautophagy is not the way that glycogen is delivered to lysosomes, overturning a long held belief. In collaboration Dr. Raben and Dr. Takikita, E Richard and G Drouillard from France explored the consequences of substrate deprivation by using shRNA for glycogenin or for glycogen synthase in culture or delivering shRNA for glycogen synthase intramuscularly in mice using an AAV vector to Pompe mice. Both methods of blocking glycogen synthesis reduced glycogen accumulation in muscle cells. More recently, studies with Pompe mice lacking glycogen synthetase have been made and are being studied to see if the method of substrate deprivation can prevent the clinical manifestations of Pompe disease. We have also converted mouse fast twitch to slow twitch fibers by crossing Pompe mice to mice that are transgenic for PGC1alpha in an attempt to permit effective ERT and in parallel we are trying to achieve the same end chemically by using SIRT1 agonists such as resveratrol and related compounds.